Efficient production of clerodane and ent-kaurane diterpenes through truncated artificial pathways in Escherichia coli

The clerodane and ent-kaurane diterpenoids are two typical categories of diterpenoid natural products with complicated polycyclic carbon skeletons and significant pharmacological activities. Despite exciting advances in organic chemistry, access to these skeletons is still highly challenging. Using synthetic biology to engineer microbes provides an innovative alternative to bypass synthetic challenges. In this study, we constructed two truncated artificial pathways to efficiently produce terpentetriene and ent-kaurene, two representative clerodane and ent-kaurane diterpenes, in Escherichia coli. Both pathways depend on the exogenous addition of isoprenoid alcohol to reinforce the supply of IPP and DMAPP via two sequential phosphorylation reactions. Optimization of these constructs provided terpentetriene and ent-kaurene titers of 66 ± 4 mg/L and 113 ± 7 mg/L, respectively, in shake-flask fermentation. The truncated pathways to overproduce clerodane and ent-kaurane skeletons outlined here may provide an attractive route to prepare other privileged diterpene scaffolds.


General experimental procedures.
Restriction enzymes were purchased from Takara (Beijing, China). PCR primers were synthesized by Sangon Biotech Co., Ltd (Shanghai, China). DNA sequencing was performed by Sangon Biotech Co., Ltd (Shanghai, China). Phanta Super-Fidelity DNA Polymerase, ClonExpress II One Step Cloning Kit, DNA gel extraction and plasmid extraction kits were purchased from Vazyme Biotech (Nanjing, China). Other chemicals and biochemicals were purchased from standard commercial sources. 1 H and 13 C NMR experiments were run on a Bruker Avance NEO at 400 MHz for 1 H and 100 MHz for 13 C nuclei. HPLC was performed on an Agilent 1260 Infinity with an Agilent Poroshell 120 EC-C18 column (50 mm × 4.6 mm, 2.7 μm).

Plasmids construction.
The plasmids, primers, and gene sequences used in this study are listed in Tables S1, S2 and S4, respectively. Plasmids were constructed using homologous recombination [1]. The phoN, ipk, bjks, citB, and citI genes were codon optimized and synthesized by General biosystems (Anhui, China). The ggdps, tdps, and ttes genes were amplified from Kitasatosporia griseola DSM 43859. The plasmid pLD10001 containing phoN and ipk was synthesized by General Biosystems (Anhui, China). The ecdps gene was cloned from Streptomyces sp. NRRL S-1813. The idi gene was cloned from E. coli.

Plasmid pLD10003 was constructed by inserting ggdps and idi into the NcoI and
HindIII sites in the pRSFDuet-1 vector. The PCR products of crtB and crtI were assembled into the linearized vector of pRSFDuet-1 resulting in pRSFDuet-crtB-crtI.
The crtE and idi genes were cloned into NcoI/BamHI and NdeI/XhoI sites of the pRSFDuet-1 resulting in pRSFDuet-crtE-idi. The crtB-crtI and crtE-idi fragments including their T7 promoters were assembled into the linearized vector pRSFDuet-1 resulting in pLD10004. The tdps gene was cloned into the PstI/HindIII sites of pRSFDuet-1 vector to construct plasmid pLD10005. Then the ttes gene was cloned into S3 the NdeI/XhoI sites of pLD10005 to construct plasmid pLD10006. After pLD10006 construction, the tdps-ttes fragment including their T7 promoters was cloned and inserted into pLD10003 between the EcoRI and KpnI sites, resulting in plasmid pLD10007. The ggdps-idi-tdps-ttes fragment including their T7 promoters was inserted into pLD10001 between EcoRV and KpnI sites, resulting in plasmid pLD10010. The bjks and ecdps genes were cloned into the EcoRV/XhoI and NdeI sites of the pRSFDuet-1 vector to construct the plasmid pLD10008. The PCR product bjks-ecdps including their T7 promoters was inserted into pLD10003 resulting in pLD10009. The ggdpsbjks-ecdps-idi fragment including their T7 promoters was inserted into pLD10001 between EcoRV and KpnI sites, resulting in plasmid pLD10011. All plasmids were verified by DNA sequencing.

Fermentation of lycopene in engineered strains.
Each plasmid containing lycopene expression genes was transformed into E. coli BL21 (DE3) and plated on LB ager supplemented with ampicillin (100 μg/mL) and kanamycin (50 μg/mL) for incubation overnight at 37 °C. Colonies were picked the following day and used to inoculate 5 mL LB with ampicillin (100 μg/mL) and kanamycin (50 μg/mL) for incubation overnight at 37 °C. A 250 μL seed culture was then used to inoculate 50 mL of LB with 2% glycerol and ampicillin (100 μg/mL) and kanamycin (50 μg/mL) added and grown at 37 °C at 230 rpm. Until the culture reached OD600 ≈ 0.6, flasks were cooled using an ice bath. Then, IPTG and DMAA/ISO were added to a final concentration of 0.1 mM and 6 mM, respectively. The cells were grown around 3 days at 18 °C with shaking at 200 rpm.

Identification and isolation of terpentetriene and ent-kaurene.
Two plasmids of pLD10001 and pLD10007 were transformed into E. coli BL21 (DE3) to create strain DL10004. Then, this strain was fermented in LB medium supplied with 1% glycerol and shaking at 37 °C with a speed of 230 rpm until reaching an OD600 of 0.6. Then, the mixture was cooled to 4 °C, followed by addition of 6 mM ISO/DMAA S4 3:1 and 0.1 mM isopropyl β-ᴅ-1-thiogalactopyranoside (IPTG) inducer. After a 3-day fermentation at 18 °C with a shaking speed of 200 rpm, the cells were harvested by centrifugation at 3750 rpm for 20 min at 4 °C and extracted with acetone for three times.
After centrifugation, the supernatant was combined and concentrated under vacuum to obtain the crude extract, which was then passed through a silica gel column with isometric elution of pure petroleum ether to afford 45 mg terpentetriene. The pure sample was subjected to 1 H and 13 C NMR experiments.
Two plasmids of pLD10001 and pLD10009 were transformed into E. coli BL21 (DE3) to create strain DL10006. Then, the strain DL10006 was fermented in LB medium at 37 °C supplemented with 1% glycerol until reaching an OD600 of 0.6. Then, the mixture was cooled to 4 °C using an ice bath, followed by the addition of 6 mM ISO/DMAA 3:1 and 0.1 mM IPTG. After a 3-day fermentation at 18 °C with a shaking speed at 200 rpm, the cells were harvested by centrifugation at 3750 rpm for 20 min at 4 °C. The cell pellets were then extracted with acetone for three times. After centrifugation, the acetone supernatant was combined and concentrated under vacuum to obtain the crude extract, which was then passed through a silica gel column with isometric elution of pure petroleum ether to afford 90 mg ent-kaurene. The pure sample was subjected to 1 H and 13 C NMR experiments. Their chemical structures were determined by analysis the 1 H and 13 C NMR spectra and compared with reported data [2,3].     Table S1: Details of primers used in this study.